Skin-specific full skin models, also called in vitro skin equivalents, can be used in particular in dermatology and allergiology as a test skin in order to test substances, for example, potential medications or cosmetics, or agents such as light and heat, for their pharmacological effects, in particular irritation, toxicity, and inflammation effects, as well as their compatibility. Such a system also can be used to answer many types of immunological, histological, and molecular-biological questions. This includes, for example, studies of wound healing and of the penetration and absorption of substances. Compared with animal experiments and studies using human test subjects, the studies or tests of substances with such full skin models offer substantial advantages since the results obtained with them are more reproducible and the studies are less costly and quicker.
In recent years, mostly human cell cultures have been used as in vitro systems for the testing of raw materials and products. A further development in cell culture technology are three-dimensional, organ-like human cell structures and co-culture systems. The results obtained with these can be transferred to humans even better than the results obtained with single cell cultures. The developments by Rheinwald and Green (Rheinwald, J. G. et al., “Serial cultivation of strains of human epidermal keratinocytes: The formation of keratinizing colonies from single cells”, Cell, 6 (1975), 331-344; Green, H. et al., “Growth of cultured human epidermal cells into multiple epithelia suitable for grafting”, Proc. Nat. Acad. Sci. USA 76 (1979), 5665-5668) were the start of the cultivation of human keratinocytes and their use in burn medicine and in vitro dermatology. In the past, different models of reconstructed skin have been produced in vitro.
EP 0 197 090 B1 discloses a method for forming a skin equivalent, wherein a hydrated collagen network is produced by mixing an acidic collagen solution with contractile cells, for example, fibroblasts. After neutralizing the pH value, collagen fibrils are precipitated in the collagen network. The contractile cells attach to the collagen network and bring about its contraction, whereupon a dermis equivalent forms. By introducing punch biopsy skin samples into the collagen network, keratinocytes from the punch biopsy samples are able to grow on the surface of the dermis equivalent, whereupon a skin equivalent forms.
EP 0 285 474 B1 discloses a skin equivalent comprising a dermis equivalent obtained from collagen and fibroblasts, as well as a multilayer epidermis equivalent. The dermis equivalent is hereby inoculated with a human or animal explant, for example, a hair follicle, in order to obtain the epidermis equivalent.
EP 0 020 753 B1 describes a method for forming tissue, especially skin tissue, wherein fibroblasts are introduced into a hydrated collagen network also, and a tissue forms after the collagen network contracts. Keratinocytes previously cultivated in vitro or keratinocytes extracted from foreskin can be applied to this tissue, whereupon a skin replacement is formed.
EP 0 418 035 B1 discloses a tissue equivalent comprising a hydrated collagen network contracted with a contractile agent, such as fibroblasts, and a collagen gel that is in contact with a permeable element. The mixture of collagen and contractile agent is applied to the collagen gel, whereby the contact between collagen gel and permeable element, for example, a polycarbonate membrane, interrupts the radial or lateral contraction of the collagen network so that the network only contracts with respect to its thickness. After the dermis equivalent forms, keratinocytes can be seeded, whereupon a skin equivalent forms.
U.S. Pat. No. 5,861,153 furthermore forms a skin equivalent consisting of an epidermis equivalent on a carrier, whereby the epidermis equivalent comprises keratinocytes and induced or non-induced precursors of Langerhans cells.
The carrier may be a collagen network containing fibroblasts or dermis sections from which the epidermis has been removed, artificial membranes, a subcutaneous replacement based on collagen, or synthetic materials.
U.S. Pat. No. 4,963,489 describes stroma tissue prepared in vitro, whereby the stroma cells, for example, fibroblasts, envelop a basic network consisting of a biocompatible material, for example, cellulose. The described system can be used, for example, to produce a three-dimensional skin culture system, whereby keratinocytes and melanocytes are applied to the dermis equivalent, i.e., the three-dimensional stroma carrier matrix.
U.S. Pat. No. 5,755,814 describes a skin model system that can be used both as an in vitro test system and for therapeutic purposes. The system comprises a three-dimensional, networked matrix of insoluble collagen that contains fibroblasts and stratified layers of differentiated epidermis cells, whereby an epidermis cell layer is in direct contact with the surface of the collagen matrix. The networking of the matrix can be achieved both using a thermal treatment with water removal, as well as by chemical means, for example, with carbodiimide.
U.S. Pat. No. 5,882,248 describes a method for determining the effects of chemical substances or agents on a human skin model system according to U.S. Pat. No. 5,755,814. The interaction between the skin model system and the substances to be tested is determined with the help of the release of substances by cells of the skin model system as well as with the effects on metabolism, proliferation, differentiation, and reorganization of these cells.
WO 95/10600 furthermore describes a method with which an epidermis equivalent can be obtained. This epidermis equivalent can be used for pharmaceutical and/or cosmetic sun tanning tests.